Abstract Roughly 2,000 new drugs and chemicals are commercialized each year successfully clearing research and development (R&D) hurdles to meet the safety requirements of regulators such as the U.S. Food and Drug Administration. These successes, however, are the exception rather than the rule making it advantageous for R&D programs to have methods in place that ensure unsafe compounds ?fail fast? well before entering time and resource intensive animal or human testing. In vitro toxicity assays have assumed this early screening role but lack the ability to assess the contribution of potentially toxic metabolites that the body produces from otherwise safe parent chemicals. The integration of metabolic profiling into current toxicity screening platforms is therefore crucial to chemical risk, exposure, and drug safety assessments. The cytochrome P450 (CYP) family is one of the most important families of enzymes involved in human metabolism, metabolizing about 75% of all xenobiotics and drugs in the liver. Integrating the function of CYPs and other metabolic families (e.g., UGTs) in drug metabolism and pharmacokinetics (DMPK) are key to increasing the safety of drugs as well as speeding their development. Current approaches used by pharmaceutical companies and contract research organizations rely on cultured hepatocytes, which co-activate multiple enzymatic pathways in a single integrated platform. Although this method mimics liver physiology, there are several drawbacks including limited incubation time and metabolite production, the need to statistically evaluate significant background metabolomes, and complex quality control challenges inherent in cell handling techniques. Zymtronix has developed a novel, cell-free technology that offers increased metabolite production, enzyme specific metabolomes with little background, and improved quality control compared to cell-based systems, allowing for reduced cost and ease-of-use. This Phase I project will generate a proof-of-concept product for a Tox21-compatible, high-throughput (HTP) metabolic processing unit built on a proprietary enzyme-anchoring technology. First, we will adapt our existing technology to a functionalized 96-well microplate lid, and then optimize and characterize the newly engineered scaffold using CYP3A4, CYP2B6, and UGT1A6 model enzymes. Finally, we will use liquid chromatography?mass spectrometry analysis to demonstrate the conversion of six model compounds by the enzyme trio (individually and in combination) and compare the metabolome with that produced by pooled human liver microsomes. The project will prepare us for future Phase II research, in which we will explore a larger number of chemical substrates with an expanded set of CYPs/UGTs to determine a critical set of metabolic enzymes that produce a diverse and physiologically relevant metabolome. The product will be constructed in 96-well microplate format to facilitate HTP robotic handling and integration with downstream cell-based assays.